Targeting a KH-domain protein with RNA decoys

Abstract

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the aCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to aCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for aCP decoy action. Because aCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of aCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.

Shared Stabilization Functions of Pyrimidine-Rich Determinants in the Erythroid 15-lipoxygenase and α-globin mRNAs

“…The expression of the decoys in the appropriate compartment of the transfected cells was confirmed by reverse transcription-PCR (data not shown) (22). When cotransfected with U1-R7␣1, the half-life of ␣ 2R mRNA is shortened from 12 h to 9.0 h (P ϭ 0.006), while the control vector expressing the U1 framework RNA without the inserted decoy cassette has no appreciable effect (Fig.…”

“…A reciprocal study was next attempted to detect DICE activity. In this study ␣CP function endogenous to the RRL was blocked by a high-affinity ␣CP decoy RNA (22). The rationale for the study and the predicted outcomes, based on previous reports, are summarized in Fig.…”

“…4A. A synthetic decoy RNA aptamer, R7␣1, generated by SELEX (38), was embedded within a VA1 RNA framework (22). The highly structured VA1 RNA serves as an effective and stable RNA framework for the decoy cassette (for details, see reference 16).…”

“…The linkage of mRNA stabilization to ␣CP was tested by blocking ␣CP function in trans with a set of decoy RNAs. The U1-R7␣1 vector expresses high levels of the R7␣1 aptamer in the nucleus, while VA1-R7␣1 packages the same R7␣1 aptamer in a cytoplasmic (VA1 RNA) delivery framework (22). The U1-R7␣1 and VA1-R7␣1 decoy RNAs bind ␣CP with equivalent affinities (Fig.…”

Effects of local mRNA structure on posttranscriptional gene silencing

“…The 95-nt R7␣1 mRNA was transcribed (T7 MEGAshortscript; Ambion) from a DNA template generated by PCR from the pVA1-R7␣1 vector (36) with the primer pair 5Ј-TAATACGACTCACTATAGGAGAGGATACTACACGT-3Ј (T7 promoter underlined) and 5Ј-GCCAAGCTTCTCTACATGC-3Ј. Capping and purification was carried out as above, and the final sequence of R7␣1 was verified as 5Ј-GAGAGGATACTACACGTGGAGTGACCTTCTCAACTTTATATTCCCTTTACCCTTC-CCCCAAGGCACTCCATTGCATGTAGAGAAGCTTGGCG-3Ј by using the fmol DNA Cycle Sequencing System as directed by the manufacturer (Promega).…”